Neurotrophic factors are natural proteins, found in the nervous system or in non-nerve tissues innervated by the nervous system, whose function is to promote the survival and maintain the phenotypic differentiation of nerve and/or glial cells (Varon and Bunge (1978) Ann. Rev. Neurosc. 1:327; Thoenen and Edgar (1985) Science 229:238). In vivo studies have shown that a variety of endogenous and exogenous neurotrophic factors exhibit a trophic effect on neuronal cells after ischemic, hypoxic, or other disease-induced damage. Examples of specific neurotrophic factors include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), and the insulin-like growth factors I and II (IGF-I, IGF-II).
Some neurotrophic factors, such as bFGF and CNTF, are thought to have broad trophic effects, promoting survival or providing a maintenance function for many different types of neuronal cells. Other neurotrophic factors have a narrower, more specific trophic effect and promote survival of fewer types of cells. For example, in the peripheral nervous system NGF promotes neuronal survival and axonal extension of certain specific neuronal cells types such as sensory and sympathetic neurons (Ebendal et al. (1984) Cellular and Molecular Biology of Neuronal Development, Ch. 15, ed. Black, I.B.). However, in the central nervous system (CNS), NGF also supports the survival of cholinergic neurons in the basal forebrain complex (Whittemore et al. (1987) Brain Res. Rev. 12:439-464).
BDNF, a basic protein of molecular weight 12,300, supports some sensory neurons that do not respond to NGF (Barde et al. (1982) EMBO J. 1:549-553 and Hofer and Barde (1988) Nature 331:261-262). Neurotrophin 3 (NT3) supports survival of dorsal root ganglion neurons and proprioceptive neurons in the trigeminal mesencephalic nucleus. CNTF, a protein of about molecular weight 23,000, supports ciliary ganglion neurons in the parasympathetic nervous system, sympathetic neurons, dorsal root ganglion neurons in the sensory nervous system, and motor neurons in the CNS (Kandel et al. (1991) Principles of Neural Science, 3rd Ed., Elsevier Science Publishing Co., Inc., N.Y.).
Some neurotrophic factors constitute a family of neurotrophic factors characterized by about 50% amino acid homology. One such family is the NGF/BDNF family, which includes BDNF, NGF, NT3 and NT4 (Hohn et al. WO 91/03569; U.S. patent application Ser. No. 07/680,681 now abandoned). Both NGF and BDNF are apparently synthesized as larger precursor forms which are then processed, by proteolytic cleavages, to produce the mature neurotrophic factor (Edwards et al. (1986) Nature 319:784; Leibrock et al. (1989) Nature 319:149). There is a significant similarity in amino acid sequences between mature NGFs and mature BDNF, including the relative position of all six cysteine amino acid residues, which is identical in mature NGFs and BDNF from all species examined (Leibrock et al. (1989) supra.). See FIG. 2, comparing and emphasizing the similarities of human forms of BDNF (SEQ ID NO:3) and NGF (SEQ ID NO:4). This suggests that the three-dimensional structures of the mature proteins, as determined by the location of the disulfide bonds, are similar. The mature NGFs and BDNF proteins also share a basic isoelectric point (pI).
NGF is a neurotrophic factor at least for cholinergic neurons in the basal forebrain (Hefti and Will (1987) J. Neural Transm. [Suppl] (AUSTRIA) 24:309). The functional inactivation and degeneration of the basal forebrain cholinergic neurons responsive to NGF in the course of Alzheimer's disease is thought to be the proximate cause of the cognitive and memory deficits associated with that disease (Hefti and Will (1987) supra.). NGF has been shown to prevent the degeneration and restore the function of basal forebrain cholinergic neurons in animal models related to Alzheimer's disease, and on this basis has been proposed as a treatment to prevent the degeneration and restore the function of these neurons in Alzheimer's disease (Williams et al. (1986) Proc. Natl. Acad. Sci. USA 83:9231; Hefti (1986) J. Neuroscience 6:2155; Kromer (1987) Science 235:214; Fischer et al. (1987) Nature 329:65).
BDNF is a neurotrophic factor for sensory neurons in the peripheral nervous system (Barde (1989) Neuron 2:1525). On this basis, BDNF may prove useful for the treatment of the loss of sensation associated with damage to sensory nerve cells that occurs in various peripheral neuropathies (Schaumberg et al. (1983) in Disorders of Peripheral Nerves, F. A. Davis Co., Philadelphia, Pa.).
In order for a particular neurotrophic factor to be potentially useful in treating nerve damage, it must be available in sufficient quantity to be used as a pharmaceutical treatment. Also, since neurotrophic factors are proteins, it is desirable to administer to human patients only the human form of the protein, to avoid an immunological response to a foreign protein. Since neurotrophic factors are typically present in vanishingly small amounts in tissues (e.g., Hofer and Barde (1988) Nature 331:261; Lin et al. (1989) Science 246:1023) and since human tissues are not readily available for extraction, it would be inconvenient to prepare pharmaceutical quantities of human neurotrophic factors directly from human tissues. As an alternative, it is desirable to use the isolated human gene for neurotrophic factor in a recombinant expression system to produce potentially unlimited amounts of the human protein.
Mature, biologically-active neurotrophic factors can be produced when human or animal neurotrophic factor genes are expressed in eukaryotic cell expression systems (e.g., Edwards et al. (1988) Molec. Cell. Biol. 8:2456). In such systems, the full-length neurotrophic factor precursor is first synthesized and then proteolytically processed to produce mature neurotrophic factor which is correctly folded 3-dimensionally and is fully biologically active. However, eukaryotic cell expression systems often produce relatively low yields of protein per gram of cells and are relatively expensive to use in manufacturing.
In contrast, expression systems that use prokaryotic cells, such as bacteria, generally yield relatively large amounts of expressed protein per gram of cells and are relatively inexpensive to use in manufacturing. However, obtaining biologically active bacterially-expressed neurotrophic factor has been a major hurdle in this field. Bacteria are not able to correctly process precursor proteins, such as the precursor protein for NGF, by making appropriate proteolytic cleavages in order to produce the correct smaller mature protein. Therefore, to produce mature neurotrophic factor in bacteria, it is necessary to express only that portion of the DNA sequence encoding the mature protein and not that for the larger precursor form. When this was done in E. coli, relatively large amounts of the mature human NGF protein were produced (see, e.g., Iwai et al. (1986) Chem. Parm. Bull. 34:4724; Dicou et al. (1989) J. Neurosci. Res. 22:13; European Patent Application 121,338). Unfortunately, the bacterially-expressed protein had no apparent biological activity.
Bacterial production of recombinant mammalian proteins often result in biologically inactive proteins forming inclusion bodies. This necessitates separating the inclusion bodies from other cell components, and solubilizing the inclusion bodies to unfold the protein (Spalding (1991) Biotechnology 9:229). The likely reason for this lack of biological activity is that the mature protein is unable to assume spontaneously the correct 3-dimensional structure and form the correct intramolecular disulfide bonding pattern required for full biological activity. Processing includes the separation and solubilization of the inclusion bodies, unfolding the protein, then refolding the protein into the correct biologically active tertiary structure. However, during refolding, the protein may reaggregate, reducing the yield of active protein and further complicating the purification process (Spalding (1991) supra).
Protocols for unfolding and refolding NGF have been described (e.g., European Patent Application 336,324; U.S. Pat. Nos. 4,511,503 and 4,620,948). However, these protocols have serious deficiencies. Many protocols use exposure of NGF to high pH to break incorrectly formed disulfide bonds followed by exposure to lower pH to allow formation of correct intramolecular disulfide bonds. The exposure of NGF to high pH is known to result in extensive modification of the protein, including elimination of amine side chains in glutamine and asparagine (of which there are 7 in mature human NGF), and extensive chemical alteration of asparagine-glycine, asparagine-serine, and asparagine-threonine adjacent pairs (of which there are 2 in mature human NGF). In addition to these chemical modifications, the refolding procedure appeared to restore only approximately one-tenth of the biological activity of NGF. Although numerous protocols for refolding and renaturing proteins that do not involve harsh conditions exist, no such procedure has been applied successfully to NGF. For a general review of refolding procedures, see Kohno (1990) Methods Enzymol. 185:187.
Various methods have been used to improve recovery of biologically active proteins produced in a bacterial expression system. One method for cleaving incorrectly formed disulfide bonds is the use of S-sulfonated proteins obtained by sulfitolysis (U.S. Pat. No. 4,421,685; Gonzalez and Damodaran (1990) J. Agric. Food Chem. 38:149; European Patent Application 361,830). The addition of sulfite to a protein initially cleaves the disulfide bonds exposed to the solution, resulting in the formation of one S-SO.sub.3.sup.- derivative and one free SH group for each disulfide bond cleaved. In the presence of an oxidizing agent, the free SH groups are oxidized back to disulfide, which is again cleaved by the sulfite present in the system. The reaction cycle repeats itself until all the disulfide bonds and the sulfhydryl groups in the protein are converted to cys-SO.sub.3.sup.-. Generally, this allows most proteins to be fully solubilized (European Patent Application 361,830).
Another method to improve the recovery of biologically active protein from bacterial expression systems includes the use of polyethylene glycol (PEG) in the refolding mixture. It has been proposed that the addition of PEG prevents protein aggregation resulting from the association of hydrophobic intermediates in the refolding pathway. Cleland et al. (1990) Biotechnology 8:1274 and (1992) J. Biol. Chem. 267:13327, reported improved recovery of biologically active bovine carbonic anhydrase B (CAB) with the addition of PEG during the refolding process. The concentration of PEG required to achieve an increase in the recovery of active protein was twice the total protein concentration, and required PEG with molecular weights of 1000-8000 (Cleland et al. (1992) supra).
A bacterial expression system for producing NGF is disclosed in Canadian Patent No. 1,220,736 and U.S. Pat. No. 5,169,762. However, no procedures for refolding the expressed protein are presented. A procedure for producing large quantities of biologically active recombinant NGF suitable for pharmaceutical use is described in U.S. patent application Ser. No. 08/087,912 filed Jul. 6, 1993 now abandoned by Collins et al., entitled: Production of Biologically Active, Recombinant Members of the NGF/BDNF Family of Neurotrophic Proteins. The protein is exposed to a denaturant, such as guanidine hydrochloride or urea, and sufficient reducing agent, such as .beta.-mercaptoethanol, dithiothreitol, or cysteine, to denature the protein, disrupt noncovalent interactions, and reduce disulfide bonds. The free thiols present in the reduced protein are then oxidized, and the protein allowed to form the correct disulfide bonds. The refolding mixture preferably contained up to 25% PEG 200 or 300.
While the procedure described in U.S. patent application Ser. No. 08/087,912 now abandoned achieves improved yields of biologically active NGF, the need remains for more efficient means for refolding NGF. The bacterial production of recombinant proteins results in biologically inactive proteins found as inclusion bodies within the bacterial cell. There is a need for improved processing methods for separating the inclusion bodies from other cell components and solubilizing the inclusion bodies to unfold the protein. Further, there is a need for improved methods for breaking incorrectly formed disulfide bonds and refolding the protein into the correct tertiary structure required for maximum yield of fully active protein while decreasing chemical modification of the protein.
The present disclosure presents an extended and improved method for producing bacterially-expressed biologically active members of the NGF/BDNF family of neurotrophic factors, including the first use of the process of sulfitolysis to solubilize and chemically modify a neurotrophic factor.